Cannabinoids, represented by (−)-Δ9-Tetrahydrocannabinol (Δ9-THC), are the active components of Cannabis Sativa, and known to exhibit behavioral and psychotropic effects, and possess therapeutic properties in a variety of areas such as the central nervous system, the cardiovascular system, the immune system and endocrine system.
Most of the effects of cannabinoids are due to interaction with specific high-affinity receptors. Presently, two cannabinoid receptors, CB1 and CB2, have been identified. Characterization of these receptors has been made possible by the development of specific synthetic ligands such as the agonists WIN 55212-2 (aminoalkyl indole) and CP 55,940 (non-classic cannabinoid).
The CB1 cannabinoid receptor has been primarily detected in the central nervous system (CNS) and to a lesser extent in certain peripheral tissues, such as, pituitary gland, immune cells, reproductive organs, gastrointestinal tissues, superior cervical ganglion, heart, lung, retina, urinary bladder and adrenal gland. The central distribution pattern of CB1 receptors accounts for several prominent pharmacological properties of cannabinoids, such as impairing cognition and memory and alternating the control of motor function, and mediating the psychotropic effects and other neurobehavioral effects of cannabinoids. Activation of CB1 receptor has been linked to a number of therapeutic indications seen in cannabinoids, such as analgesia, management of emesis, anxiety, feeding behavior, neuro-protection, movement disorder, glaucoma, cancer and cardiovascular diseases.
Conversely, the CB2 cannabinoid receptor does not appear to be expressed within the CNS but is primarily expressed within immune system. A high abundance of CB2 receptors have been detected in human tonsils, leukocytes, and spleen. In human leukocytes, CB2 receptors were found with particularly high concentration in B-cells, natural killer cells and macrophage. The high abundance of cannabinoid receptor CB2 subtype in the immune system suggest that the CB2 receptor could be the most likely cannabinoid receptor that mediates the immunomodulatory effects of cannabinoids. The immune modulatory effects of cannabinoids are very broad, including altering immune cell proliferation and function, altering antibody formation and altering cytokine production.
The discovery of cannabinoid receptors was followed by the identification of their endogenous ligands. So far, five of endogenous eicosanoids found in mammalian brain and certain other tissues have been identified to resemble the pharmacological activities of Δ9-THC and bind to the cannabinoid receptors. These endogenous cannabinergic eicosanoids include anandamide (arachidonoylethanolamide), 2-arachidonoylglycerol (2-AG), homo-γ-linolenoylethanolamide and docosatetraenoylethanolamide, and noladin ether. These compounds, collectively named as endocannabinoids, are synthesized by cells on demand, and released upon depolarization-induction. Endocannabinoids are generally produced by stimulus-dependent cleavage of membrane phospholipid precursors through two steps of enzymatic reactions: 1) generation of N-acyl-phosphatidylethanolamine (NAPE) via N-acylation of phosphatidylethanolamine (cephalin) by a N-acyltransferase, and 2) formation of N-acylethylamine from cleavage of NAPE by a N-acyl-phosphatidyl-ethanolamine-specific phospholipase D (NAPE-PLD). Of these endocannabinoids, the most investigated to date have been anandamide and 2-AG. There are indications that both anandamide and 2-AG can serve as neuromodulators or neurotransmitters.
In general, the endocannabinoids have been found to be somewhat less potent than Δ9-THC. Despite having a rapid onset of action, the magnitude and duration of action of these molecules are relatively short, presumably because of a rapid inactivation process, which is necessary to terminate their biological effects. The inactivation of anandamide appears to be a two-step process. It is first relocated from the extracellular side by carrier-facilitated reuptake and passive diffusion into cells, where it is then degraded by hydrolysis, catalyzed by the enzyme fatty-acid-amide-hydrolase (FAAH).
A high-affinity anandamide transporter has been characterized in rat cortical neurons and in astrocytes, but has not yet been isolated or cloned. This “anandamide transporter” appears to be a lipid uptake protein similar to, but distinct from, the prostaglandin uptake system. It was found that anandamide transport constitutes the rate-limiting step in the biological inactivation of anandamide, both in vitro and in vivo. Compound N-(4-hydroxyphenyl)-arachidonylamide (AM404) has been demonstrated to selectively and potently inhibit such transport, without binding to cannabinoid receptors or affecting anandamide-hydrolysis. Inhibitor AM404 was shown to enhance the receptor-mediated effects of anandamide, suggesting a potential for therapeutic intervention through transporter blockade.
Post reuptake, anandamide and 2-AG are disposed by a membrane-bound protein, fatty acid amide hydrolase (FAAH). FAAH was cloned in 1996. Its crystal structure was recently determined. FAAH is pH-dependent, selective and sensitive to an irreversible inhibitor PMSF (phenylmethylsulfonylfluoride).
Alternatively, an amidase, which is distinct from FAAH but also hydrolyzing anandamide and other N-acylethanolamines at acidic pH, was identified in human megakaryoblastic cells and rat organs such as lung and spleen. As for the 2-AG hydrolysis, in addition to the known monoacylglycerol (MAG) lipase, other esterases and FAAH may be involved. There are clear correspondences between brain levels of anandamide following pretreatment with FAAH inhibitors and pharmacological activity, which infer FAAH inhibitors as indirect sources for cannabinoid receptor activation.
It is also known that cyclo-oxygenase (COX) is the enzyme that catalyses the conversion of arachidonic acid to prostaglandins. To date, two isoforms are known, COX1 and COX2. COX1 is constitutively expressed in most tissues and COX2 is expressed in inflamed and neoplastic tissues. Experimental studies have shown that cyclo-oxygenase 2 (COX2) is involved in tumour development and progression. Hence, selective inhibitors of COX2 (coxibs) blocking tumour growth may be used as potential anticancer agents. In addition, inhibitors of COX2 also may be used as anti-inflammatory agents.
The CB1 and CB2 receptors, and the anandamide transporter as well as the enzymes involved in the biosynthesis, function and degradation of endocannabinoids have emerged as novel targets for therapeutic interventions. Two CB1 agonists, Marinol® and Cesamet®, have been marketed as controlled/non-controlled medications for treatment of anorexia seen in AIDS and cancer patients, and emesis associated with chemotherapy respectively. Currently, a CB1 inverse agonist (SR141716A) is in clinic trial for treatment of eating disorders. Although agonists of cannabinoid receptors exhibit a broad spectrum of pharmacological effects, the psychotropic properties of CB1 agonists have strongly limited the development of cannabinoid-based medications. A number of approaches have been taken to overcome these limitations, such as finding CB1 inverse agonists, CB2 selective agonists, water-soluble CB1/CB2 agonists, non-psychoactive cannabinoids, co-administration of CB1 agonists with other known medications as an effect-enhancer and agents indirect stimulating CB1 and CB2.
Synthetic analogues of the endocannabinoids commonly exhibit different functions to the endogenous cannabinoid system, including binding to the cannabinoid receptors CB1 and CB2 with enhanced affinity or bio-stability, inhibiting the enzyme FAAH or inhibiting the “anandamide transporter” as well as regulating the enzymes involved in bio-synthesis of endocannabinoids. These distinct features are rarely co-presented in one molecule. Inhibitors of FAAH, MAG lipase or anandamide transporter have the ability of indirectly stimulating the CB1 and CB2 receptors by maintaining or elevating de novo level of anandamide, therefore, serving as indirect agents to activate the cannabinoid receptors. Endocannabinoid analogues possessing the aforementioned features may exhibit fewer side effects than the ligands directly activating CB1 receptors, and provide novel therapeutic approaches to the disease states associated with the endocannabinoid system, such as pain, psychomotor disorders, multiple sclerosis, emesis, anxiety, feeding behaviors, glaucoma, neuro-degradation, cardiovascular disease and immune malfunction.